DNA tagged microparticles

ABSTRACT

A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 13/608,962, filed on Sep. 10, 2012, entitled “Bio-ThreatMicroparticle Simulants”, now U.S. Pat. No. 8,835,178, issued on Sep.16, 2014, which is a continuation of U.S. Pat. No. 8,293,535, issued onOct. 23, 2012, which is a non-provisional application claiming priorityto U.S. Provisional Patent Application No. 61/257,242 filed Nov. 2,2009, from all of which priority is claimed, and the entire contents anddisclosures of which are hereby incorporated by reference herein.

The United States Government has rights in this invention pursuant toContract No. DE-AC52-07NA27344 between the United States Department ofEnergy and Lawrence Livermore National Security, LLC for the operationof Lawrence Livermore National Laboratory.

FIELD OF THE INVENTION

The present invention relates to testing and more particularly to safeand effective stimulants for testing.

BACKGROUND

U.S. Pat. No. 7,781,224 issued Aug. 24, 2001 to Sue I. Martin et altitled “Safe Biodegradable Fluorescent Particles,” assigned to LawrenceLivermore, National Security, LLC., provides the following state oftechnology information:

-   -   The present invention provides a “safe” fluorescent particle for        a variety of applications. The particle comprises a        non-biological, biodegradable carrier and natural fluorophores        encapsulated in the non-biological, biodegradable carrier. In        some embodiments the particle is used as a simulant for        mimicking the fluorescence properties of microorganisms.        However, the particle need not mimic the fluorescent        characteristics of a microorganism, rather it can be        incorporated into one or more natural fluorophores as a means        for fluorescence detection. Single or combinations of        fluorophores are encapsulated to produce a desired fluorescent        effect such as particles that fluoresce at 370 nm maxima. The        particles can therefore be tuned to the excitation wavelength of        a fluorescence detector.    -   One application for these particles is their use in aerosol        studies, such as large scale air dispersal to track particulate        migration over vast areas, or for urban particle dispersion        studies. Currently, researchers performing these studies rely on        air dispersion models and gas tracer tests to determine the        movement and flow of aerosols in urban environments such as in        cities—around and through occupied buildings—because “safe”        particles are not available. These particles would provide those        safety benefits. Furthermore, these particles could be designed        with the appropriate density and perhaps shape of a        microorganism to mimic the aerodynamic movement of a        microorganism.    -   An example of aerosol study is described in the article, “An        examination of the urban dispersion curves derived from the St.        Louis dispersion study” by Akula Venkatram in Atmospheric        Environment 39 (2005) 3813-3822, which describes the St. Louis        study conducted over the period 1963-1965. The study consisted        of a series of 26 daytime and 16 evening experiments in which        fluorescent zinc cadmium sulfide particles were released near        ground level at two different locations under a variety of        meteorological conditions. During the first year of the        experiments, the release was at ground level in a relatively        open area in a park located west of the downtown area. In the        second year, the tracer was released from the top of a        three-story building surrounded by trees and similar buildings.        The main downtown area, consisting of buildings with an average        height of 40 m, was about 5 km away from both release        locations.” The disclosure of the article, “An examination of        the urban dispersion curves derived from the St. Louis        dispersion study” by Akula Venkatram in Atmospheric Environment        39 (2005) 3813-3822 is incorporated herein by this reference.    -   Another example of aerosol study is described in the article,        “Use of Salt Lake City URBAN 2000 Field Data to Evaluate the        Urban Hazard Prediction Assessment Capability (HPAC) Dispersion        Model” by Joseph c. Chang in Journal Of Applied Meteorology        pages 485-501 (2005), which provides the following background        about the study: “The potential impacts of the atmospheric        release of chemical, biological, radiological, and nuclear        (CBRN) or other hazardous materials are of increasing concern.        Hazardous releases can occur due to accidents, such as the        release of toxic industrial chemicals in Bhopal, India, in 1984        (e.g., Sharan et al. 1996) and the Chernobyl nuclear power plant        disaster in the Ukraine in 1986 (e.g., Puhakka et al. 1990).        They can also occur as an unintentional result of military        actions, such as the U.S. destruction of rockets with chemical        warheads at Khamisiyah, Iraq, after the 1991 Gulf War        (Winkenwerder 2002). More recently, terrorist incidents in urban        settings, such as the events on 11 Sep. 2001 in New York City,        N.Y., and Washington, D.C., and military conflicts dramatically        raise concerns for the possibility of mass casualties.” The        disclosure of the article, “Use of Salt Lake City URBAN 2000        Field Data to Evaluate the Urban Hazard Prediction Assessment        Capability (HPAC) Dispersion Model” by Joseph c. Chang in        Journal Of Applied Meteorology pages 485-501 (2005) is        incorporated herein by this reference.

SUMMARY

Features and advantages of the present invention will become apparentfrom the following description. Applicants are providing thisdescription, which includes drawings and examples of specificembodiments, to give a broad representation of the invention. Variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this descriptionand by practice of the invention. The scope of the invention is notintended to be limited to the particular forms disclosed and theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

Conducting atmospheric releases in order to challenge detector sensornetworks poses unique challenges. With the increasing use of biosensorsfor the detection of threat agents there is a growing need for auniversal biosimulant. The universal simulant needs to have severalproperties to allow for real world evaluation of biodetector andcollection networks. First, the material must be able to be collectedand trigger the detector. Second, the simulant must be safe to releasein an environment where workers and the public will be exposed to thematerial. Third, the material must be able to have controllable aerosolproperties, such as charge and physical or aerodynamic size. Theinvention details a low cost, safe, individually designed particle forthe use in testing biosensor networks.

The present invention provides a microsphere/microparticle simulantcomprising a carrier and DNA encapsulated in the carrier. The presentinvention has all of the desired properties for a universal simulant.Not only will the universal simulant be able to test and evaluate singledetectors it will be optimal for the validation of atmospheric releasemodels with multiple sensors. Currently a release study with a singlesimulant requires a costly experiment for a single release location. Ifmultiple release locations are desired multiple studies must beconducted to allow each release location and transport pathway to beuniquely identified. By using Applicants' new DNA containing biosimulantmultiple releases can occur simultaneously. This is accomplished bymodifying the unique DNA sequence for the release material. Using uniqueDNA allows for a near limitless variety of unique particle identifiers.

The microsphere simulant can be used as challenge-test standards fordetermining sensitivity of detection technologies. The microspheresimulant can be used for large-scale air current deployments or testsfor determining the movement and distribution of particles in urbanenvironments. The microsphere simulant can be labeled to distinguishbetween “test” microspheres and background microorganisms/organicparticles. The microsphere simulant can be used as a calibrationstandard for bio-detectors. The microsphere simulant can be used totrain personnel to operate bio-detectors. Surface properties, such ashydrophobicity and surface charge, can be tuned/altered for variousapplications. The microsphere simulant provides a universal simulantthat can be used for field aerosol studies, mock biowarfare training,training for rapid assessment of bioweapons labs, calibrating detectionequipment, and other uses.

The invention is susceptible to modifications and alternative forms.Specific embodiments are shown by way of example. It is to be understoodthat the invention is not limited to the particular forms disclosed. Theinvention covers all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theclaims.

Other aspects and embodiments of the present invention will becomeapparent from the following detailed description, which, when taken inconjunction with the drawings, illustrate by way of example theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of the specification, illustrate specific embodiments of theinvention and, together with the general description of the inventiongiven above, and the detailed description of the specific embodiments,serve to explain the principles of the invention.

FIG. 1 illustrates a microsphere containing DNA strand andGluconodelta-lactone (GDL).

FIG. 2 illustrates a microsphere containing antibody trigger and GDL.

FIG. 3 illustrates a microsphere containing DNA, antibody trigger,fluorophore and GDL.

FIG. 4 illustrates a microsphere containing material to control particletransit properties.

FIG. 5 illustrates a microsphere containing multiple additives combinedin a single microsphere.

FIG. 6 illustrates how multiple microspheres, each containing uniqueDNA, enables for simultaneous releases during a single event.

FIG. 7 illustrates a method, according to one embodiment.

FIG. 8 illustrates data associated with the release of microspherescomprising a DNA barcode from a single location, according to oneembodiment.

FIG. 9 illustrates data associated with the release of two sets ofmicrospheres from two locations, where each set of microspherescomprises a unique DNA barcode, according to one embodiment.

DETAILED DESCRIPTION

Referring to the drawings, to the following detailed description, and toincorporated materials, detailed information about the invention isprovided including the description of specific embodiments. The detaileddescription serves to explain the principles of the invention and is notmeant to limit the inventive concepts claimed herein. The invention issusceptible to modifications and alternative forms. The invention is notlimited to the particular forms disclosed. The invention covers allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the claims. Further, particularfeatures described herein can be used in combination with otherdescribed features in each of the various possible combinations andpermutations.

Unless otherwise specifically defined herein, all terms are to be giventheir broadest possible interpretation including meanings implied fromthe specification as well as meanings understood by those skilled in theart and/or as defined in dictionaries, treatises, etc.

It must also be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless otherwise specified.

As used herein, the term “about” when combined with a value refers toplus and minus 10% of the reference value. For example, a length ofabout 1000 nm refers to a length of 1000 nm±100 nm, a temperature ofabout 50° C. refers to a temperature of 50° C.±5° C., etc.

The January/February 2002 issue of Science & Technology Review, in anarticle titled “Rapid Field Detection of Biological Agents,” describestwo systems to rapidly detect and identify biological agents, includingpathogens such as anthrax and plague. The systems are the HandheldAdvanced Nucleic Acid Analyzer (HANAA) and the Autonomous PathogenDetection System (APDS). About the size of a brick, the HANAAbiodetection system can be held in one hand and weighs less than akilogram. The system was designed for emergency response groups, such asfirefighters and police, who are often first on the scene at sites wherebioterrorism may have occurred. Each handheld system can test foursamples at once-either the same test on four different samples or fourdifferent tests on the same sample. HANAA can provide results in lessthan 30 minutes, compared with the hours to days that regular laboratorytests typically take. To detect the DNA in a sample, a synthesized DNAprobe tagged with a fluorescent dye is introduced into the sample beforeit is inserted into the heater chamber. Each probe is designed to attachto a specific organism, such as anthrax or plague. Thus, the operatormust have an idea of what substances might be involved. “The systemdoesn't test for all unknowns,” says Langlois. “A responder has todecide what kinds of pathogens to test for ahead of time and set up thesystem accordingly.” If that organism is present in the sample, theprobe attaches to its DNA, which is then amplified during the PCRprocess, releasing the fluorescent tag. HANAA measures the sample'sfluorescence and the presence (or absence) of the targeted organism.Whereas HANAA can be handcarried to sites at which an attack issuspected to have happened, the APDS is stationed in one place forcontinuous monitoring and is designed to work much like a smokedetector, but for pathogens. When fully developed, the APDS could beplaced in a large area such as an airport, a stadium, or a conferencehall. The system will sample the air around the clock and sound an alarmif pathogens are detected. The disclosure of the article titled “RapidField Detection of Biological Agents,” in the January/February 2002issue of Science & Technology Review is incorporated herein by thisreference.

The October 2004 issue of Science & Technology Review, in an articletitled “Detecting Bioaerosols When Time is of the Essence,” states thatLivermore researchers received seed funding from the Laboratory DirectedResearch and Development Program to develop an instrument that countersbioterrorism by providing a rapid early warning system for pathogens,such as anthrax. That instrument, the Autonomous Pathogen DetectionSystem (APDS), is now ready for deployment to better protect the publicfrom a bioaerosol attack, and the development team has been honored witha 2004 R&D 100 Award. In September 2003, APDS passed a series ofpathogen exposure tests at a high-containment laboratory at the DugwayProving Ground in Utah. In these trials, the system clearly demonstratedthat it could detect real pathogens and confirm the identifications witha fully automated second assay method. APDS units were also deployed atthe Albuquerque Airport in New Mexico and at a Washington, DC, Metrostation, where they provided continuous monitoring for up to seven days,unattended. The system can be adapted for situations where environmentalor clinical pathogens require monitoring. For example. APDS could testfor mold or fungal spores in buildings or for the airborne spread ofcontagious materials in hospitals. It also could identify diseaseoutbreaks in livestock transport centers or feedlots. The disclosure ofthe article titled “Detecting Bioaerosols When Time is of the Essence,”in the October 2004 issue of Science & Technology Review is incorporatedherein by this reference.

The evaluation of different biofluorescence detectors as tools to detectbiological attack is currently difficult due to the lack of a single,common standard with which to compare the different instruments.Biological organism stimulants present substantial drawbacks in thatthey are difficult to transport and aerosolize without damaging them,exposure to them presents a health risk, and they have a tendency toagglomerate, which makes their aerosolization difficult to performreliably. Furthermore, they have a short shelf life, they are notconveniently disposable, their use requires extensive training, anyequipment exposed to them requires bleach or otherbactericides/sporicides for cleaning, they are difficult to manufacture,and many aspects of their growth and handling affect their final state.Therefore, biological organisms are not optimal evaluation, calibration,and training standards for biofluorescence instruments. They are,however, fluorescent in the precise manner of a microorganism(obviously), which is ultimately necessary for a test agent orsurrogate.

Conducting atmospheric releases in order to challenge detector sensornetworks poses unique challenges. With the increasing use of biosensorsfor the detection of threat agents there is a growing need for auniversal biosimulant. The universal simulant needs to possess severalproperties to allow for real world evaluation of biodetector andcollection networks. First, the material must be able to be collectedand trigger the detector. Second, the simulant must be safe to releasein an environment where workers and the public will be exposed to thematerial. Third, the material must be able to have controllable aerosolproperties, such as charge and physical or aerodynamic size.

The present invention incorporates all of the desired properties. Notonly will the universal simulant be able to test and evaluate singledetectors it will be optimal for the validation of atmospheric releasemodels with multiple sensors. Currently a model study with a singlesimulant requires a costly study for a single release location. Ifmultiple locations are desired multiple studies must be conducted. Byusing Applicants' new DNA containing food safe material multiplereleases can occur simultaneously. This is accomplished by modifying theunique DNA sequence for the release material. Using unique DNA allowsfor a near limitless variety of unique particle codes.

Microsphere Composition

According to one embodiment, a microsphere comprises a carrier and atleast one DNA barcode encapsulated in the carrier. As used herein invarious approaches, a microsphere may also be referred to as amicrosphere simulant and/or a microparticle.

In some approaches, the morphology of the carrier may be spherical orsubstantially spherical, non-spherical (e.g. elliptical, tubular, etc.),irregular etc. In other approaches, the size of carrier may be betweenabout 1 nm to about 100 μm.

In preferred approaches, the carrier may comprise a non-toxic material.In some approaches, the carrier may comprise a material that isnon-toxic to at least one of: humans, animals (domesticated animals,wild animals, etc.), the environment, etc. Suitable carrier materialsnon-toxic to humans and/or animals may include, but are not limited to,a non-mutagenic material; a non-carcinogenic material; anon-radiological material; a material that is not harmful, hazardous,poisonous or otherwise deleterious to the health of humans and/oranimals; a biocompatible ingestible material (e.g. a material suitableand/or intended to be swallowed/ingested by humans and/or animalswithout eliciting any undesirable local or systemic effects in humansand/or animals upon ingestion), etc. Additionally, suitable carriermaterials non-toxic to the environment may include materials that arenon-hazardous, nonpoisonous or otherwise do not have anegative/deleterious impact on an ecosystem (e.g. an interacting systemof a biological community including but not limited to plants, mammals,reptiles, fish, microbial communities, etc.) and its non-livingenvironmental surroundings (e.g. soil, water, air, man-made structures,etc.). For instance, in some approaches, suitable materials non-toxic tothe environment may be materials that are biodegradable (e.g. subject todegradation over a period of time due to exposure by biological and/orenvironmental conditions such as sunlight, fluids, temperature,naturally occurring microorganisms such as bacteria, etc.).

In one particular approach the non-toxic carrier material may comprise abiological material and/or a non-biological material. In anotherapproaches, the non-toxic carrier material may comprise a carbohydratesuch as maltodextrin, glucono-delta-lactone, or other suitablecarbohydrate as would be understood by one having skill in the art uponreading the present disclosure.

In yet another approach, the non-toxic carrier material may be at leastone of a food, a food additive, a FDA approved food additive, a coloradditive, and a FDA approved color additive. The term “food” may referto any synthetic and/or naturally occurring raw, cooked, or processededible/ingestible substance suitable and/or intended, in whole or inpart, for human and/or animal consumption, in some embodiments.Additionally, a food additive may refer to any synthetic and/ornaturally occurring substance which may result, or may reasonably beexpected to result, directly or indirectly, in becoming a component offood and/or otherwise affecting the characteristics of food, in variousembodiments. Further, in numerous embodiments, a color additive mayrefer to a dye, pigment or other such substance made by a syntheticprocess, extracted, isolated, or otherwise derived, with or withoutintermediate or final change of identity, from a vegetable, animal,mineral, or other source and that, when added or applied to a food, iscapable (alone or through reaction with another substance) of impartinga color thereto. Moreover, a FDA approved additive, whether a foodadditive or a color additive, may refer, in some embodiments, to anadditive subject to approval by the United States Food and DrugAdministration (FDA) as being generally regarded as safe (e.g. notharmful under the intended conditions of use).

In a further approach, the non-toxic carrier material may be a materialselected from a group consisting of: a carbohydrate, a food, a foodadditive, a FDA approved food additive, a color additive, a FDA approvedcolor additive, a protein, a vitamin, talc, silica, an antacid, and acombination thereof.

As noted above, the microsphere comprises at least one DNA barcodeencapsulated in the carrier. As used herein, a DNA barcode refers to asynthetically produced nucleic acid. In some approaches, the at leastone DNA barcode encapsulated in the carrier may comprise between about80 to about 150 bases.

In various approaches, the DNA barcode may comprise a known sequence.For example, in one embodiment, the sequence of the DNA barcode maycomprise a signature associated with threat agents such as pathogens,hazardous and/or toxic chemical, biological, radiological and nuclearmaterials, etc. and other threat agents as would be understood by onehaving ordinary skill in the art upon reading the present disclosure.However, in other embodiments, the sequence of the DNA barcode may beexclusive of signatures associated with threat agents. In moreembodiments, the sequence of the DNA barcode may be exclusive ofsignatures associated with a material/substance/particle that isnaturally occurring in the environment, such as naturally occurringspores. Conversely, in even more embodiments, the sequence of the DNAmay include signatures associated with a materials/substances/particlesnaturally that is occurring in the environment. In still moreembodiments, the at least one DNA barcode may comprise a sequenceconfigured to activate/trigger a hand held diagnostic test.

In exemplary approaches, the at least one DNA barcode comprises a uniquesequence. This is advantageous where a first carrier encapsulating afirst DNA barcode and a second carrier encapsulating a second DNAbarcode are released in the same physical location and/or locationshaving at least partially overlapping perimeters and/or areas. Forinstance, collection and/or subsequent identification of the releasedcarriers as being one of the first carrier or one of the second carriermay be facilitated where the first DNA barcode and the second DNAbarcode each have unique sequences, i.e. the sequence associated withthe first DNA barcode is different that the sequence associated with thesecond DNA barcode. For DNA barcode's comprising 100 bases, there areapproximately 10⁶⁰ unique combinations.

In further approaches, the microsphere may also comprise an additionalmaterial (a material other than the at least one DNA barcode), where theadditional material is encapsulated in the carrier. The additionalmaterial may be selected from a group consisting of: a fluorophore, aprotein, an immunoassay trigger, an isotope marker, a chemicalsignature, a sunscreen material, and combinations thereof, in variousapproaches.

In one exemplary approach, the additional material may comprise afluorophore such as tryptophan. In another approach, the additionalmaterial may be configured to alter one or more properties of thecarrier including but not limited to solubility, density, charge, size,morphology, etc. and other such properties as would be understood by onehaving skill in the art upon reading the present disclosure.

Microsphere Production

Several methods can be used to produce microsphere particles from liquidsolution. The methods discussed herein focus on aerosolizing thesolution and drying the resulting aerosol with a desiccant dryer. Thetest results discussed focus on the use of an ink jet printer to producethe initial droplets of biosimulant material. This method is used toproduce a liquid droplet with reproducible size distributions. Othermethods to aerosolize the material are also possible to generate theparticles. Other aerosol production methods include salter and collisionnebulizers for solution aersolization. The resulting liquid droplets aredried with a desiccant dryer and collected in a chamber or particleimpactor. Large quantities of particles may be dried by other methodssuch as a spray dryers or low humidity counter flow apparatus.

FIG. 7 depicts a method 700 for forming a microsphere, such as thosedescribed herein and shown in the other FIGS., according to an exemplaryembodiment. The method 700 may be carried out in any desiredenvironment. Moreover, more or less operations than those shown in FIG.7 may be included in method 700, according to various embodiments. Itshould also be noted that any of the aforementioned features may be usedin any of the embodiments described in accordance with this method andothers disclosed herein.

As shown in FIG. 7, the method 700 includes providing a carriercomprising a non-toxic material. See operation 702. In some approaches,the non-toxic material may be non-toxic to at least one of: humans,animals (domesticated animals, wild animals, etc.), the environment,etc. In another approach the non-toxic material may be a biologicalmaterial and/or a non-biological material. In yet another approach, thenon-toxic material may be selected from a group consisting of: acarbohydrate, a food, a food additive, a FDA approved food additive, acolor additive, a FDA approved color additive, a protein, a vitamin,talc, silica, an antacid, and a combination thereof.

The production of the microspheres with FDA approved food product allowsfor the ability of ingestion of the material when it is aerosolized, inexemplary approaches. Water soluble food material such as GDL posesminimal risk for inhalation and ease of sample handling in theproduction process. By using water based material no organic solventsare needed greatly reducing any potential health and safety issues.

As also shown in FIG. 7, the method 700 includes providing at least oneDNA barcode. See operation 704. In one approach, the at least one DNAbarcode encapsulated in the carrier may comprise between about 80 toabout 150 bases. In another approach, the DNA barcode may comprise asequence that includes or excludes signatures associated with threatagents, and/or that includes or excludes signatures associated with amaterial/substance/particle that is naturally occurring in theenvironment. In yet another approach, the at least one DNA barcode maycomprise a sequence configured to activate/trigger a hand helddiagnostic test.

The method 700 additionally includes encapsulating the at least one DNAbarcode in the carrier. See operation 706. In one approach, at least twoDNA barcodes may be encapsulated in the carrier. In various approaches,these at least two encapsulated DNA barcodes may comprise sequences thatare partially or completely the same. In other approaches, these atleast two encapsulated DNA barcodes may comprise sequences that arepartially or completely different.

In some embodiments, the method 700 may further include forming thecarrier and the at least one DNA barcode encapsulated therein intodroplets, and aerosolizing the droplets.

In another embodiment, the method 700 may optionally include providingan additional material and encapsulating the additional material in thecarrier. The additional material may be selected from a group consistingof: a fluorophore, a protein, an immunoassay trigger, an isotope marker,a chemical signature, a sunscreen material, and combinations thereof, inexemplary approaches.

In further approaches, the composition of the carrier material mayaltered in order to tune one or more of the following properties of thecarrier: density, morphology, solubility, size, charge, number of copiesof the DNA barcode in the carrier, etc. or other such property as wouldbe understood by one having skill in the art upon reading the presentdisclosure.

In yet another embodiment, the method 700 may further include providinga second carrier comprising a second non-toxic material, andencapsulating at least one unique DNA barcode in the second carrier. Insome approaches, the first and second non-toxic materials associatedwith the first and second carriers, respectively, may be the same ordifferent. In other approaches, the at least one unique DNA barcodeencapsulated in the second carrier may be the same or different than theat least one unique DNA barcode encapsulated in the first carrier.

Any of the methods, microspheres/microparticles, systems, etc. describedabove, taken individually or in combination, in whole or in part, may beincluded in or used to make one or more systems, products, etc. Inaddition, any of the features presented herein may be combined in anycombination to create various embodiments, any of which fall within thescope of the present invention. Following are several examples ofgeneral and specific embodiments.

EXAMPLES

The illustrative, non-limiting examples described below refer tomicrospheres 100 that include a carrier 101 comprising a non-toxic foodproduct, and DNA 102 encapsulated in the carrier 101, as shown inFIG. 1. As an option, the microsphere 100 of FIG. 1 may be implementedin conjunction with features from any other embodiment listed herein,such as those described with reference to the other FIGS. Of course, themicrosphere 100 and others presented herein may be used in variousapplications and/or in permutations which may or may not be specificallydescribed in the illustrative embodiments listed herein. Further, themicrosphere 100 presented in FIG. 1 may be used in any desiredenvironment.

As discussed above, microspheres 100 may be produced by combining DNA102 a carrier 101 material. The microspheres are produced by dissolvingthe carrier material and DNA in an aqueous solution and aerosolizing theresulting solution. The material is aerosolized to break the solutioninto small droplets. The size of the initial droplet and initialconcentration of the solution dictates the final particle size. Largerdroplets and higher concentration solutions will produce larger particlesizes. The aerosols are dried with a drying apparatus and themicrospheres are collected in a collection chamber.

The specific experiments/examples described below refer to the formationof a microsphere via dissolution of a carrier material 101 comprising atleast 10% Glucono-delta-lactone (GDL), and trigger DNA 102 in an aqueoussolution. Furthermore, in the experiments described below, the triggerDNA 102 consists of ˜100 DNA bases of thermotoga maritime. With 100 DNAbases and 4 possible substitutions for each base the maximum theoreticaltotal number of unique combinations is 4^100 (1.6 e 60). The resultingsolution is divided up into droplets with an inkjet print-head or otheraerosol production method and the water is removed to produce thesimulant. The resulting particle is a safe, size selectable biosimulantcontaining DNA. By changing the concentration of the GDL or droplet sizethe size of the dried biosimulant can be selected.

It is important to note that the examples provided herein are not meantto be limiting in any way, but rather solely provide illustrativeembodiments of the present invention.

Example 1 Production of Microspheres with a FDA Food Additive and DNA

Glucono-delta-lactone (GDL), a FDA approved kosher certified foodadditive, was used as the carrier material for the microsphereproduction. Aqueous solutions of 15% GDL were combined with knownamounts of DNA. The aqueous solution was aerosolized with an inkjetprinter and the resulting particles were dried with a desiccant dryer.The dried particles were collected on a particle impactor. The resultsfrom all production tests at this solution concentration show a sizedistribution centered at ˜1.75 microns. The particles produced containedDNA in the target size range and had a spherical morphology. The sizewas measured with an aerosol particle sizer and the spherical morphologywas confirmed with SEM images.

The mean microsphere size increases linearly with concentration of GDL.This is important, as it shows that the GDL microsphere size may betailored to the application of interest. As we are interested inproducing microspheres between 1 and 5 μm in diameter, we chose 15% GDLas the ideal starting solution to produce microspheres with the desiredproperties. It is also possible to alter the microsphere size byaltering the initial aqueous droplet size. The resulting particle is asafe, size selectable biosimulant containing DNA.

The microsphere simulant 100 can be used as challenge-test standards fordetermining sensitivity of detection technologies. The microspheresimulant 100 can be used for large-scale air current deployments ortests for determining the movement and distribution of particles inurban environments. The microsphere simulant 100 can be labeled todistinguish between “test” microspheres and backgroundmicroorganisms/organic particles. The microsphere simulant 100 can beused as a calibration standard for bio-detectors. The microspheresimulant 100 can be used to train personnel to operate biodetectors.Surface properties, such as hydrophobicity and surface charge, can betuned/altered for various applications. The microsphere simulant 100provides a universal simulant that can be used for field aerosolstudies, mock biowarfare training, training for rapid assessment ofbioweapons labs, calibrating detection equipment and other uses.

As illustrated in FIG. 2, DNA and antibody trigger 103 is combined withcarrier material to produce microspheres stimulants. The microspherestimulant is also generally designated by the reference numeral 100 inFIG. 2. The incorporation of the antibody trigger 103 allows thebiosimulant 100 to trigger immuno assay detectors. The antibody trigger103 is added to an aqueous solution of food safe material (currentlyGDL) or other carrier 101. The resulting solution is aerosolized anddried to form antibody trigger microspheres. It is critical to selectthe antibody trigger and antibody to take into account any health riskswhen the particle is released. Many natural antibody triggers such asproteins can cause allergic reactions and this must be taken intoaccount when the trigger material is selected. Antibodies can beproduced for a large range of target material ranging from explosives tobovine serum albumin. This diversity of antibodies allows for a largerange of potential antibody trigger chemicals. The concentration ofantibody trigger and carrier can be easily modified to change both thesize of the final particle tuned to select the desired properties for agiven experiment.

The microsphere simulant 100 illustrated in FIG. 2 can be used forlarge-scale air current deployments or tests for determining themovement and distribution of particles in urban environments. Themicrosphere simulant 100 can be labeled to distinguish between “test”microspheres and background microorganisms/organic particles. Themicrosphere simulant 100 can be used as a calibration standard forbio-detectors. The microsphere simulant 100 can be used to trainpersonnel to operate bio-detectors. Surface properties, such ashydrophobicity and surface charge, can be tuned/altered for variousapplications. The microsphere simulant 100 provides a universal simulantthat can be used for field aerosol studies, mock biowarfare training,training for rapid assessment of bioweapons labs, calibrating detectionequipment, and other uses.

As illustrated in FIG. 3, DNA 102 and antibody triggers 103 andfluorescent molecules 104 are combined with a carrier 101 to producemicrospheres stimulants 100. Antibody trigger fluorescent molecules andDNA are added to an aqueous solution of food safe material (currentlyGDL) or other carrier. By adding multiple fluorescent molecules a uniqueand tunable fluorescence signal can be achieved. The microspheresimulant 100 illustrated in FIG. 3 can be used for large-scale aircurrent deployments or tests for determining the movement anddistribution of particles in urban environments. The microspheresimulant 100 can be labeled to distinguish between “test” microspheresand background microorganisms/organic particles. The microspheresimulant 100 can be used as a calibration standard for bio-detectors.The microsphere simulant 100 can be used to train personnel to operatebiodetectors. Surface properties, such as hydrophobicity and surfacecharge, can be tuned/altered for various applications. The microspheresimulant 100 provides a universal simulant that can be used for fieldaerosol studies, mock biowarfare training, training for rapid assessmentof bioweapons labs, calibrating detection equipment, and other uses.

As illustrated in FIG. 4, materials to alter the particle transportproperties 105 are combined with a carrier to produce microspheres 100.Additives are added to a solution of carrier material and the resultingsolution is used to produce microspheres. Properties such as charge anddensity greatly alter aerosol transport properties. By adding materialto alter these properties a highly tunable particle can be produced tosimulate a natural particle or a threat agent. Being able to reproducethe transport properties of aerosols will allow for more detailedstudies of atmospheric release of pollutants and threat materials.

As illustrated in FIG. 5, microspheres with DNA 102, antibody triggers103, fluorophore 104, and materials 105 to control particle transitproperties are combined with a carrier to produce microspheres 100.Antibody trigger, fluorescent molecules, additives to control transportproperties and DNA are added to an aqueous solution of food safematerial (currently GDL) or other carrier. The resulting solution isconverted into microspheres.

Multiple varieties of DNA containing microspheres FIG. 1 are producedand simultaneously released to test bio detector networks. Currenttesting is limited to a small number of testing agents. By using theunique properties and DNA signature for test particles, multiplesimultaneous test release can be achieved. This allows for great costsavings and rapid incorporation of real word data into modelingsimulations.

Example 2

Glucono-delta-Lactone (GDL) a FDA approved kosher certified foodadditive was used as the carrier material for the microsphereproduction. Aqueous solutions of 15% GDL were combined with knownamounts of DNA. Two sequences of DNA from thermotoga maritime wereincorporated into two sets of microsphere particles. The aqueoussolution was aerosolized with an inkjet printer. The resulting aerosolwas collected and analyzed with PCR. The experiments showed that theresulting aerosol droplets can be identified with PCR.

The trigger DNA consists of ˜100 DNA bases of thermotoga maritime. With˜100 DNA bases and 4 possible substitutions for each base the maximumtheoretical total number of unique combinations is 4¹⁰⁰ (1.6 e 60).

Applications and Uses

Embodiments of the present invention may be used in a wide variety ofapplications including but not limited to those described below.

Aerosol Transport Studies

In various approaches, embodiments of the present invention may be usedfor aerosol transport studies in indoor environments. One of the mostoverlooked threats to human health is poor indoor air quality. Variousair pollutants exist indoors, including biological pollutants (molds,bacteria, viruses, pollen, animal dander, dust mites, etc.), secondhandsmoke, combustion pollutants, and other chemicals (formaldehyde,asbestos, radon, etc.). These contaminants “pool” in spaces withinadequate ventilation. As a result, the quality of indoor environmentscan suffer, with detrimental effects on human health or even abuilding's structural integrity.

Moreover, poor heating and/or air conditioning (HVAC) systems in indoorventilation environments may also affect building occupants' comfort andhealth. For instance, building occupants in one quadrant often may betoo hot while others located in another quadrant may be too cold.

Conventional products used to track particulate migration and/orvalidate air transport models, such as natural and genetically modifiedspores, are typically expensive to produce and face significantregulatory and public perception barriers to release in public places.Additionally, once released, these spores must be removed, making therelease site temporarily unavailable (up to several days) and limitingadditional testing. For instance, after a traditional release usingBacillus subtilis or Bacillus thuringensis spores, the area must bedecontaminated prior to a second release, otherwise, there is no way todifferentiate whether the detected spores were from the first or secondrelease.

Embodiments disclosed herein overcome the aforementioned drawbacks byproviding microspheres comprising a biodegradable and/or non-toxic (e.g.safe) simulant material made with non-biological DNA barcodes, and whichcan track and quantify indoor airflow. These disclosed microspheres(also referred to as microparticles) may simulate the aerosolscomprising the air around us, and thus may help identify flow patterns.For example, the microspheres disclosed herein may be released in afacility/structure with one or more air-handling units to identify whichof the units are functioning properly (e.g. collect and condition theair as desired). Such facilities may include, but are not limited to, asubway station, a train station, an airport, a corporate building, anoffice, convention centers, a warehouse, an apartment complex, a school,a home, an airplane, a submarine, or other similar facility/structure aswould be understood by one having skill in the art upon reading thepresent disclosure.

Further, the potential for creating unique DNA barcodes is virtuallyunlimited, thus allowing for simultaneous and repeated releases of themicrospheres comprising unique DNA barcodes, which dramatically reducesthe costs associated with conducting source attribution testing forcontaminants. For example a plurality of microspheres each of whichcomprise DNA barcodes different from one another can be released in thesame and/or different areas of a facility and still be identifiedthrough their DNA bar codes. Moreover, use of microspheres comprisingthese DNA barcodes encapsulated in a carrier allow multiple releases tooccur in a short time frame, in contrast to other conventional methodsthat require a cleanup of the facility between tests. Accordingly, useof the microspheres disclosed herein to conduct aerosol testing (e.g. totrack particulate migration over a vast area for indoor testing) allowsfor the validation of models and aerosol detector locations that havepreviously been unobtainable.

According to one embodiment, a method of using the microspheresdisclosed herein for indoor aerosol transport studies may includereleasing a plurality of microspheres from a release location,collecting and/or detecting the microspheres at one or more collectionlocations, and analyzing the collected microspheres to identify the DNAbarcodes encapsulated therein. In some approaches, a few, some, or allof the released microspheres may comprise DNA barcodes that are the sameor different relative to each other. It is important to note that such amethod is not limited to releasing microspheres at a single releaselocation. For instance, in other approaches, microspheres may bereleased at two or more release locations, where some or all or thereleased microsphere may comprise the same or different DNA barcodesrelative to each other.

In various approaches, the one or more collections locations maycorrespond to existing locations already having existing collectionmechanisms/devices/detectors able to collect and/or detect themicrospheres, such as a filter, a swab, etc. or other suchmechanism/device as would be understood by one having skill in the artupon reading the present disclosure. In other approaches, the collectionlocations may be selected by a user and correspond to locations at whicha user chooses to place/situate and/or install a collectionmechanism/device/detector, etc. These collection locations may also belocated at varying distances from the release location. For instance,microspheres may be detected several meters from the release point tomore than 100 meters from the release point.

In further approaches, the plurality of collected microsphere may beanalyzed in a polymerase chain reaction (PCR). PCR primers may bedesigned to only anneal to the DNA barcode of interest (e.g. the DNAbarcode associated with the released microspheres) in order to avoiddetecting any contaminant that is in the air, in preferred approaches.In even more approaches, the detected microspheres may be subject toadditional analysis to determine, for example, the size (e.g. theaerodynamic diameter) of each microsphere, the number of copies of DNAbarcode encapsulated in each microsphere, etc.

Reference is made to FIG. 8 which illustrates an exemplary experimentinvolving the tracking and quantification of particulate migration in anindoor environment using the microspheres disclosed herein. As shown inFIG. 8, a plurality of microspheres 802 comprising a carrier and a DNAbarcode encapsulated in the carrier were released at the releaselocation 802. In this exemplary experiment, each of the microspherescomprise the same DNA barcode.

As also shown in FIG. 8, the microspheres traveled varying distancesfrom the release location 802 and were collected at four dry filterunits (DFU) 806, each of which drew 800 L/min over two polyesterfilters. The collected microspheres were then processed and analyzed byPCR. The resulting particle count was based on the quantitative PCRanalysis of the particles collected on the filters.

Reference is now made to FIG. 9, which illustrates another exemplaryexperiment involving the simultaneous release of a first set and asecond set of microspheres from two different release locations, wherethe first set of microspheres comprise a DNA barcode that is differentfrom the DNA barcodes associated with second set of microspheres. Forexample, as shown in FIG. 9, the first set of microspheres comprise theDNA barcode labeled 431 and the second set of microspheres comprise theDNA barcode labeled 359. The first set of microspheres and the secondset of microspheres were simultaneously released at a first releaselocation 902 and a second release location 904, respectively. After thesimultaneous release, the microspheres were collected on filters 906 andanalyzed by PCR to identify which of the collected microspherescontained the DNA barcode 431 and which contained the DNA barcode 359.

As illustrated in the chart in FIG. 9, the microspheres comprising theDNA barcode 359 decreased in concentration as the distance from therelease point increased. The microspheres comprising the DNA barcode 431test particles, which were released and collected at 90 degrees to thesample collection layout 908, also decreased in concentration withdistance.

The exemplary experiment shown in FIG. 9 clearly highlights the value ofusing microspheres with unique DNA barcodes for determining airflowbased on different release locations, especially as compared toconventional products for airflow testing. For instance, where aconventional product is released from multiple locations, it isimpossible to identify which of the detected particles (associated withthe product) originated from a particular release location, as there istypically no distinguishing feature amongst the released particles.

A comparison between conventional aerosol transport testing materialsand the microspheres comprising DNA barcodes described herein isprovided in the table below.

GE Genetically Microspheres Visolite Traditional modified comprisingLeak SF₆ spores/ spore DNA Detection Polymer Industrial Commercial (Noton the barcodes System Microspheres Gas pesticide market) Minimum 0 DaysDays to Days to Days to Days to time weeks weeks weeks weeks betweenreleases Number of Unlimited 3 Limited 1 Limited Unlimited uniqueparticles Safety Safe Only Hazardous Environmentally Found in Unknownimproved unfriendly nature; for (powerful possible industrial greenhousegas) contamination air handlers Ease of Easy Limited Limited Easy EasyUnlikely safety approval for aerosol Diameter .001-100 n/a 1-10 ~3 ×10⁻⁴ 0.5-4 0.5-4 of test particle (μm) End-to-end $ $ $$ $ $$$ $$$$ costfor multiple releasesAs shown in the above table, the microspheres comprising DNA barcodes,such as those disclosed herein, offer several advantages over theconventional aerosol transport testing materials. For example, Visolite,produced by GE, has limited types of test materials and is notautomatically approved for release in occupied facilities. In addition,polymer microspheres pose an inhalation hazard when released in largequantities and have a limited number of unique particle types. Moreover,spores, such as those that are used as a pesticide, also havelimitations for release in public areas and are difficult to use forstudies that require multiple releases. Moreover still, geneticallymodified spores will require extensive and repeated approval to satisfypublic environmental concerns. Finally, the industrial gas sulfurhexafluoride (SF₆) has historically been used to study gas transport dueto its easy-to-detect nature and low toxicity; however, SF₆ is a gas anddoes not accurately reproduce aerosol transport properties.

It is important to note, that embodiments of the present invention mayalso be used for aerosol transport studies in outdoor environments, aswell. For instance, in one approach, the microspheres comprising the DNAbarcodes disclosed herein may be released from an outdoor releaselocation, and later collected and identified in order to determine thedownwind spread of the microspheres. This may be particularly useful formeteorological studies involving the study of wind and outdoor airflow.

Source Attribution

In another approach, the microspheres disclosed herein may be used bothto track the unwanted release of chemical/biological agents, and forsource attribution. For example, releasing these microspheres from smokestacks, mining operations or other industrial facilities may not onlyhelp assess the potential migration of greenhouse and other toxic gases(e.g. CO₂, NO_(x), SO_(x), CH₄, N₂O, O₃, CFCs, etc.), but also helpidentify which facilities are guilty of releasing said gases.Accordingly, companies and regulatory agencies can determine whereeffluents are likely to go, as well as the source of the effluents, byusing the microspheres disclosed herein. Characterization of the airflowand source of these effluents will not only aid in emergency responsefrom an unexpected release but can also help in false accusations ofcontamination.

Moreover, with the uniquely coded DNA, one could also apply thesedisclosed microspheres to oil and gas operations, fracking operations,etc. to better track fluid flow.

Response Training

The microspheres comprising the DNA barcodes disclosed herein may beused to evaluate and train individuals on how to respond to an incident.For instance, in one approach, these microspheres may be used to mimicsmoke or other hazardous materials in an industrial setting.Accordingly, releasing these microspheres and studying their migrationin such environments may help identify safe harbor sites and areas toavoid in an emergency. Using the microspheres in this manner would beparticular useful to identify shelter areas in a mine fire or otherfacility where indoor is the preferable or only option in an emergency.

In another approach, the microspheres comprising the DNA barcodesdisclosed herein may be used to evaluate decontamination protocols. Forexample, these microspheres may be dispersed on an individual and/or inan indoor or outdoor facility. After the clean-up/decontaminationprocedure is completed, the effectiveness of the procedure may beevaluated by determining how many of the microspheres were effectivelyand efficiently collected.

Validation of Diagnostic Tests

The microspheres comprising the DNA barcodes disclosed herein may bealso be used to validate diagnostic tests; medical, environmental andbio-hazardous containment protocols; and/or various detectors (e.g.general aerosol detectors, bio detectors, etc.) in more approaches.

Identification Applications

The microspheres comprising the DNA barcodes disclosed herein may beadditionally be used to tag an item for later identification. Forinstance, in one illustrative approach, these microsphere may be usedessentially as a barcode to identify a package or other item.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and have been described in detail herein.However, it should be understood that the invention is not intended tobe limited to the particular forms disclosed. Rather, the invention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the followingappended claims. Any of the methods, systems, devices, etc. describedabove, taken individually or in combination, in whole or in part, may beincluded in or used to make one or more systems, structures, etc. Inaddition, any of the features presented herein may be combined in anycombination to create various embodiments, any of which fall within thescope of the present invention. Following are several examples ofgeneral and specific embodiments.

What is claimed is:
 1. A product comprising: a first carrier; at leastone fluorophore encapsulated in the first carrier; and at least one DNAbarcode encapsulated in the first carrier, wherein the first carriercomprises a non-toxic material, wherein the product is in an aerosolform and is biosafe, wherein the non-toxic material is selected from agroup consisting of: a vitamin, talc, an antacid, and combinationsthereof.
 2. The product as recited in claim 1, wherein the at least oneDNA barcode has greater than or equal to about 80 bases to less than orequal to about 150 bases.
 3. The product as recited in claim 1, whereinthe first carrier has a diameter of about 1 nanometer to about 100microns, and wherein the non-toxic material of the first carrier iswater soluble.
 4. The product as recited in claim 3, wherein the firstcarrier has a diameter in a range from greater than 10 microns to lessthan or equal to 100 microns.
 5. The product as recited in claim 1,wherein the non-toxic material includes glucono-delta-lactone.
 6. Theproduct as recited in claim 1, wherein the product further comprises anadditional material configured to alter one or more properties of thecarrier, wherein the one or more properties of the carrier include adensity, a morphology, a solubility, a charge, and a size, wherein theadditional material is encapsulated in the first carrier.
 7. A system,comprising: the product as recited in claim 1; a second carriercomprising a non-toxic material; at least one fluorophore encapsulatedin the second carrier; and at least one DNA barcode encapsulated in thesecond carrier, wherein the at least one DNA barcode encapsulated in thesecond carrier is different than the at least one DNA barcodeencapsulated in the first carrier of the product.
 8. A method oftracking and/or quantifying particulate migration outside of a humanand/or animal body, comprising: releasing the product as recited inclaim 1 from one or more release locations; collecting the product atone or more collection locations; and analyzing the collected product toverify an identity, thereof.
 9. The method as recited in claim 8,wherein the product is released from at least two different locations,and wherein the product is in an aerosol form.
 10. The method as recitedin claim 9, wherein the product further comprises: a second carriercomprising a non-toxic material; and at least one DNA barcodeencapsulated in the second carrier, wherein the at least one DNA barcodeencapsulated in the second carrier is different than the at least oneDNA barcode encapsulated in the first carrier, wherein the location fromwhich the second carrier is released is different than the location fromwhich the first carrier is released.
 11. A product comprising: a firstcarrier; at least one fluorophore encapsulated in the first carrier; atleast one DNA barcode encapsulated in the first carrier; and anadditional material selected from the group consisting of: an isotopemarker, and a sunscreen composition, wherein the additional material isencapsulated in the first carrier, wherein the first carrier comprises anon-toxic material, wherein the product is in an aerosol form and isbiosafe.
 12. The product as recited in claim 11, wherein the non-toxicmaterial is selected from a group consisting of: a food, a FDA approvedfood additive, a FDA approved color additive, and combinations thereof.13. A method, comprising: providing a first carrier, wherein the firstcarrier comprises a non-toxic material; encapsulating at least oneunique DNA barcode in the first carrier; encapsulating at least onenon-toxic fluorophore in the first carrier; and aerosolizing the firstcarrier and the at least one non-toxic fluorophore and the at least oneunique DNA barcode encapsulated therein, wherein the non-toxic materialis selected from a group consisting of: a vitamin, talc, an antacid, andcombinations thereof.
 14. A method, comprising: providing a firstcarrier, wherein the first carrier comprises a non-toxic material;encapsulating at least one unique DNA barcode in the first carrier;encapsulating at least one non-toxic fluorophore in the first carrier;aerosolizing the first carrier and the at least one non-toxicfluorophore and the at least one unique DNA barcode encapsulatedtherein; and encapsulating at least one of an isotope marker and asunscreen composition in the first carrier.
 15. The method as recited inclaim 14, wherein the non-toxic material is selected from a groupconsisting of: a food, an FDA approved food additive, a kosher certifiedfood additive, a FDA approved color additive, and combinations thereof.16. The method as recited in claim 14, wherein the carrier has adiameter in a range from greater than or equal to about 1 nanometer toless than 1 micron.
 17. The method as recited in claim 14, furthercomprising: providing a second carrier, wherein the second carriercomprises a second non-toxic material; and encapsulating at least oneunique DNA barcode in the second carrier, wherein the at least oneunique DNA barcode encapsulated in the second carrier is different thanthe at least one unique DNA barcode encapsulated in the first carrier.18. The method as recited in claim 14, wherein the carrier has adiameter of greater than 15 microns to less than or equal to about 100microns.